Diagnosis of tuberculosis

ABSTRACT

The invention comprises an oligonucleotide selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6. The invention also comprises a complementary oligonucleotide of the oligonucleotide selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6, an oligonucleotide being at least 80% homogolous thereto, a truncated portion of any of the aforementioned, or a pairing of any of the aforementioned.

THIS INVENTION relates to the diagnosis of tuberculosis (TB). Moreparticularly, the invention relates to an oligonucleotide that can beused in the diagnosis of TB, to an in vitro method of diagnosing TB, andto a diagnostic kit for diagnosing TB.

Tuberculosis (TB) is one of the biggest killers among infectiousdiseases, despite the worldwide use of a live attenuated vaccine andseveral antibiotics. There are an estimated eight million new cases peryear and two million deaths annually, which are compounded by theemergence of drug resistance TB and co-infections with HIV.

Despite the enormous burden of TB, conventional approaches to diagnosiscurrently used continue to rely on tests that have major drawbacks. Manyof these tests are slow and lack both sensitivity and specificity orrequire expensive equipment and trained personnel. For example, sputumsmear microscopy is insensitive; the culture method is technicallycomplex and slow; chest radiography is non-specific, and the tuberculinskin test is imprecise, and its results are non-specific; nucleic acidamplification tests and phage display are rapid but specificity andsensitivity are low. The recently discovered nucleic acid amplificationtest called the GeneXpert-, addresses the problems of time andsensitivity but the machine required is extremely expensive.

It is accordingly an object of this invention to provide an improvedmethod of diagnosing TB, with the method being more sensitive and/ormore specific than those of which the Applicant is aware.

According to a first aspect of the invention, there is provided anoligonucleotide selected from the group comprising SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, acomplementary oligonucleotide thereof, an oligonucleotide being at least80% homologous thereto, a truncated portion of any of theaforementioned, or a pairing of any of the aforementioned.

More specifically, this aspect of the invention may comprise

-   (i) an oligonucleotide selected from the group comprising SEQ ID NO:    1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ    ID NO: 6; and/or-   (ii) a complementary oligonucleotide of an oligonucleotide selected    from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,    SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; and/or-   (iii) an oligonucleotide being at least 80% homologous to an    oligonucleotide selected from the group comprising SEQ ID NO: 1, SEQ    ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:    6; and/or-   (iv) a truncated portion of an oligonucleotide selected from the    group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID    NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; and/or-   (v) a pairing of any two oligonucleotides selected from the group    comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,    SEQ ID NO: 5, and SEQ ID NO: 6

SEQ ID NO: 1 to SEQ ID NO: 6 are as set out hereinafter, particularly inTable 1, and in the sequence listing annexed hereto.

The oligonucleotide may be a single stranded oligonucleotide or it maybe a double stranded oligonucleotide. In the preferred embodiment, theoligonucleotide is a single stranded oligonucleotide.

The oligonucleotide may be an aptamer, a truncated portion of anaptamer, or a pairing thereof, that binds specifically to aCFP-10.ESAT-6 heterodimer of a Mycobacterium strain. The aptamer mayinstead bind to a CFP-10 monomer of a Mycobacterium strain. Morespecifically, the aptamer may bind to the CFP-10.ESAT-6 heterodimer orCFP-10 monomer of the Mycobacterium tuberculosis (M. tb) strain.

By “a pairing thereof” is meant a pairing of two aptamers or a pairingof two truncated portions of aptamers or a pairing of an aptamer with atruncated portion of an aptamer; preferably, however, it refers to apairing of two aptamers.

The CFP-10.ESAT-6 heterodimer and the CFP-10 monomer are early markersof active tuberculosis (TB). Aptamers are artificial nucleic acid ornucleotide ligands that can bind any molecular or cellular target ofinterest with high affinity and specificity. Thus, in the presentinvention, it was surprisingly found that more sensitive and specificdiagnosis of TB can be achieved by means of aptamers, truncated portionsof aptamers or pairings thereof that bind specifically to theCFP-10.ESAT-6 heterodimer and the CFP-10 monomer.

According to a second aspect of the invention, there is provided anoligonucleotide which is an aptamer, a truncated portion of an aptamer,or a pairing thereof, that binds to a CFP-10.ESAT-6 heterodimer or to aCFP-10 monomer of a Mycobacterium strain.

This oligonucleotide may be selected from the group comprising SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5 and SEQ IDNO: 6, a complementary oligonucleotide thereof, an oligonucleotide beingat least 80% homogolous thereto, a truncated portion of any of theaforementioned, or a pairing of any of the aforementioned.

In accordance with a third aspect of the invention, there is provided anin vitro method of diagnosing tuberculosis (TB), said method comprising:

-   -   (a) contacting a sample taken from an individual suspected to be        infected with TB with the oligonucleotide according to the first        or second aspects of the invention in a CFP-10.ESAT-6        heterodimer binding assay; and    -   (b) determining whether or not the oligonucleotide has bound to        a CFP-10.ESAT-6 heterodimer in the sample, with binding of the        oligonucleotide to the CFP-10.ESAT-6 heterodimer thus confirming        the presence of the CFP-10.ESAT-6 heterodimer, and hence TB        infection in the sample.

In accordance with a fourth aspect of the invention, there is providedan in vitro method of diagnosing tuberculosis (TB), said methodcomprising:

-   -   (a) contacting a sample taken from an individual suspected to be        infected with TB with the oligonucleotide according to the first        or second aspects of the invention in a CFP-10 monomer binding        assay; and    -   (b) determining whether or not the oligonucleotide has bound to        a CFP-10 monomer in the sample, with binding of the        oligonucleotide to the CFP-10 monomer thus confirming the        presence of the CFP-10 monomer, and hence TB infection in the        sample.

In accordance with a fifth aspect of the invention, there is provided anucleic acid comprising an oligonucleotide sequence selected from SEQ.ID. NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5 andSEQ ID NO: 6, a complementary oligonucleotide thereof, anoligonucleotide being at least 80% homologous thereto, a truncatedportion of any of the aforementioned, or a pairing of any of theaforementioned.

It will be appreciated by a person skilled in the art of this inventionthat the binding assays may be chemiluminescent assays. In a preferredembodiment, the assay is a modified ELISA-type assay wherein theantibodies against a test molecule (CFP-10.ESAT-6 heterodimer or CFP-10monomer) are replaced with a labelled aptamer of the invention, atruncated portion thereof or a pairing thereof.

In accordance with a sixth aspect of the invention, there is provided adiagnostic kit for diagnosing tuberculosis (TB), said kit including:

-   -   (a) a device for taking a sample from an individual suspect to        be infected with TB; and    -   (b) apparatus for applying the method of diagnosing TB according        to the third or fourth aspects of the invention; and    -   (c) optionally, a positive control and/or a negative control.

It has been found that rational truncation of the originaloligonucleotide/aptamer sequences yield shorter, lower cost moleculesthat show comparable activity to the original parent sequences. Thetruncated versions of the oligonucleotide/aptamers retain those parts ofthe original (parent) oligonucleotide/aptamers which are predicted toplay a role in target-binding. The truncated aptamers show binding totarget proteins, with affinities comparable to those of the parentsequences.

It has also been found that the original full length olionucleotides canbe used in pairs for further use in a diagnostic setting.

The invention will now be described in more detail with reference to andas illustrated in the following non-limiting examples and accompanyingdrawings.

Hereinafter, the aptamers selected from the group comprising SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO:6, are also referred to as CSIR 2.2, CSIR2.9, CSIR2.11, CSIR2.15,CSIR2.19 and CSIR2.21 respectively.

In the drawings:

FIG. 1 shows neighbour joining tree for the aptamer sequences;

FIG. 2 is a graphical representation of the 24 ssDNA aptamers (CSIR 2.1to CSIR 2.24) that significantly (p<0.05) bound the CFP-10.ESAT-6 M. tbtarget protein. Statistical significant was determined by a two tailedStudent t-test and the error bars denote standard deviations ofexperiments done in triplicate. Each aptamer was tested for binding atleast twice, in independent experiments;

FIG. 3 shows binding of anti-CFP-10.ESAT-6 biotinylated ssDNA aptamersto the CFP-10.ESAT-6 heterodimer in the presence (grey shaded bargraphs) or absence (line hatched bar graphs) of anti-ESAT-6 monoclonalantibody. Error bars denote standard deviation of triplicates. Eachaptamer was tested in two independent assays;

FIG. 4 shows binding of the anti-CFP-10.ESAT-6 ssDNA aptamers to ESAT-6,CFP-10, CFP-10.ESAT-6 heterodimer, EsxGH heterodimer and HIV-1 gp120,respectively, to determine specificity. Error bars show standarddeviations of experiments done in triplicates;

FIG. 5 shows binding of the solid phase synthesised ssDNA aptamers toCFP-10.ESAT-6 heterodimer, CFP-10 and ESAT-6 monomers, respectively.Error bars denote standard deviation of triplicates. Each aptamer wastested in two independent assays;

FIG. 6 shows a specificity test for CSIR 2.11 aptamer. The specificitywas done using lysates from bacterial cultures of the auxotroph strainof M. tb. M. smegmatis, Pseudomonas, and Streptococcus. Based on astandard curve run on the plate the cut off was determined to beOD₄₅₀=0.2, with a 99% confidence interval. Error bars denote standarddeviation of triplicates. Each aptamer was tested in two independentassays;

FIG. 7 illustrates kinetic studies of 5 of the best aptamers. CFP-10attached to a CM5 chip was used to capture the respectiveanti-CFP-10.ESAT-6 aptamers. The respective aptamers were injected atdifferent concentrations at a flow rate of 10 μl/min for 5 minutes andallowed to dissociate for 10 minutes;

FIG. 8 shows folded versus unfolded aptamer binding. One batch of CSIR2.11 was refolded and the other was used directly after thawing. Therefolding step is not necessary for the binding of the aptamer to thetarget antigens. Thus, no significant difference was seen between thefolded and the unfolded aptamer. Error bars denote standard deviation oftriplicates. Each aptamer was tested in two independent assays;

FIG. 9 illustrates serial dilutions of CFP-10 on a 96 well micro-titreplate. The limit of detection for 150 nM of CSIR 2.11 is 31 ng of CFP-10M. tb protein, with an R² of 0.85. Error bars denote standard deviationof triplicates. Each aptamer was tested in two independent assays;

FIG. 10 shows the results of evaluation of clinical samples of sputumfrom patients with or without active TB using CSIR 2.11 ssDNA aptamer inan ELONA readout platform. The cut-off for positive was set at an OD₄₅₀above 0.2; denoted by the dotted line. The cut-off was determined by a99% confidence interval of a known negative sample. CSIR 3.13 aptamerisolated from the same parental library against human CD7 was used as anegative control. Error bars denote standard deviations of experimentsdone in triplicate. Each sample was tested twice in two independentstudies. The coefficient of variance between the two studies was lessthan 10% for all samples;

FIG. 11 shows, for Example 9, an evaluation of sputum samples using CSIR2.11 as a detection reagent;

FIG. 12 shows, for Example 10, that truncated aptamers show binding totarget proteins, with affinities comparable to those of the parentsequences;

FIG. 13 shows, for Example 11, aptamer sandwich data;

FIG. 14 shows, for Example 12, a comparison of CSIR 2.11 and CSIR 2.21using sputum samples;

FIG. 15 shows, for Example 13, whole bacteria ELONA.

SELECTION OF APTAMERS

Selection of aptamers has been made possible by the development of asystematic evolution of ligands by exponential enrichment (SELEX)process. The SELEX process consists of several rounds of selection ofsequences that bind to a target molecule. Each round consists of threemain stages (1) incubating the oligonucleotide library with the targetof interest, (2) separating bound targets from unbound targets using thedesired partitioning method, and (3) amplifying the bound sequences.

Both single stranded DNA (ssDNA) and RNA libraries are used in differentselections. These libraries typically consist of a random region ofnucleotides that range from 20 to 60 nucleotides, although as few as 8and as many as 220 random nucleotides can be used.

CFP-10.ESAT-6 Heterodimer

There are 23 ESAT-6-like genes in the Mtb H37Rv genome. These genes arelocated in 11 genomic loci and are named as EsxA-W. Inspection of thegenetic diversity revealed that five out of eleven cases had the Esxgenes are flanked by blocks of conserved genes. Besides the Esx genes,the other conserved regions encode proline-glutamic acid (PE) andproline-proline-glutamic acid (PPE) proteins, adenosine triphosphate(ATP) dependent chaperones of the ATPases associated with diversecellular activities (AAA) family, membrane-bound ATPases, transmembraneproteins and serine proteases, which are known as mycosins. The 11genomic regions are clustered in to five regions namely: region 1spanning the genes rv3866-rv3883c; region 2 spanning genesrv3884c-rv3895c; region 3 spanning genes rv0282-rv0292; region 4containing the genes rv3444crv3450c; and 5 containing the genesrv1782-rv1798. The genomes of Mtb H37Rv, M. bovis and M. bovis BCG havebeen compared, and various regions of difference (RD) have beenidentified. One of these regions, designated as RD1, is a 9500 bp regionthat is absent in all M. bovis BCG strains. This deletion entirelyremoves the genomic fragment from rv3872 to rv3879c. Among the lostgenes are EsxB (rv3874) and EsxA (rv3875), which respectively encodeCFP-10 and ESAT-6 proteins. This deletion is thought to be responsiblefor the primary attenuation of M. bovis to M. bovis BCG.

Function of the CFP-10 and ESAT-6

The two proteins are potent T-cell antigens recognised by over 70% oftuberculosis patients, which has led to their proposed use as adiagnostic reagent for tuberculosis in both humans and animals.

Functions of the Monomer and the Heterodimer

ESAT-6 alone or in combination with CFP-10 enhances the permeability ofartificial membranes, by disrupting the lipid bilayers and acts as acytolysin, while the exposed C-terminal region of CFP-10 may be involvedin interactions with a host cell target protein resulting instabilisation of the helical conformation.

Both proteins are important in both pathogenesis and virulence of M. tbas the CFP-10.ESAT-6 secretion system contributes to the arrest ofphagosome maturation and promotes survival of mycobacteria withinmacrophages, which provides a novel link between the CFP-10.ESAT-6secretion system and mycobacterial virulence and pathogenesis, howeverit is unclear as to whether it is ESAT-6, CFP-10 or the complex which isresponsible for the arrest of phagosome maturation.

Biochemical Pathway of the Heterodimer

Both CFP-10 and ESAT-6 are secreted by the ESAT-6 system-1 (ESX-1), adedicated secretion apparatus encoded by genes flanking EsxA and EsxB inthe extended RDI region. Among the proteins predicted to be involved inthis process are a member of the AAA-family of ATPases (Rv3868), whichmay perform chaperone-like functions by assisting in the assembly anddisassembly of protein complexes and several putative membrane proteinsor ATP binding sites, which could be involved in forming a transmembranechannel for the translocation of the effector molecules.

Why the Heterodimer is Important.

The expression characteristics of both proteins, together with theirstructural properties, have led RENSHAW, P. S., PANAGIOTIDOU, P.,WHELAN, A., GORDON, S. V., HEWINSON, R. G., WILLIAMSON, R. A. & CARR, M.D., (2002), Conclusive evidence that the major T-cell antigens of theMycobacterium tuberculosis complex ESAT-6 and CFP-10 form a tight, 1:1complex and characterization of the structural properties of ESAT-6,CFP-10, and the ESAT-6/CFP-10 complex. Implications for pathogenesis andvirulence, J Biol Chem, 277, 21598-603, to propose that the biologicallyactive form is the heterodimer. This implies that ESAT-6, without itspartner CFP-10, might not be active. The virulence of Mtb is reduced bythe knockout of either ESAT-6 or CFP-10; therefore the heterodimer isvery important in the virulence of M. tb. It has also been reported thatthe CFP-10.ESAT-6 complex acts as a signalling molecule, which is likelyto lead to the heterodimer being a key player in diagnostics.

Characterisation of the CFP-10.ESAT-6 Heterodimer

The Characteristics of the Heterodimer, Affinity of Binding, Location ofthe Heterodimer, Mechanism

Overall, the surface features of the CFP-10.ESAT-6 complex seem mostconsistent with a function based on specific binding to one or moretarget proteins. The extensive contact surface between CFP-10 and ESAT-6is essentially hydrophobic in nature and comprises about 25% of thetotal surface area of both proteins. The tight interaction between thetwo proteins in the complex appears to be primarily based on extensiveand favourable van der Waals contacts, however, two salt bridges betweenCFP-10 and ESAT-6 appear to stabilize interactions between theN-terminal end of helix-1 in CFP-10 and the C-terminal end of thecorresponding helix in ESAT-6, and between the C-terminal region ofhelix-2 in CFP-10 and the N-terminal region of the equivalent helix inESAT-6, respectively.

EXAMPLE 1 Isolation of Aptamers against CFP-10.ESAT-6 Heterodimer

Aptamer Library

The first step in the SELEX experiments was to create a pool of variantsequences from which RNA or DNA aptamers of relatively high affinity fortarget proteins could be selected. For the DNA selection a 90-mer ssDNArandomized at 49 nucleotide positions flanked by primers were customsynthesized by Integrated DNA Technologies (CA, USA). The primers were:5′-GCCTGTTGTGAGCCTCCTAAC-3′ (forward primer) and5′-GGGAGACAAGAATAAGCATG-3′ (reverse primer modified with either T7promoter region (TAATACGACTCACTATA) or a phosphate at the 3′ end). Thelibrary used was 5′-GCCTGTTGTGAGCCTCCTAAC (N49) CATGCTTATTCTTGTCTCCC-3′.

In Vitro Selection of DNA Aptamers

In the first round of selection 500 pmol of ssDNA library was used toobtain a diversity of at least 10¹⁴ molecules of random sequence. Theselection was done by a modification of the SELEX protocol in which thessDNA-protein complexes are partitioned and purified using anitrocellulose membrane. Before selection, the ssDNA library wasincubated with a nitrocellulose membrane to eliminate any membranebinders.

The ssDNA library was refolded at 95° C. for 10 minutes, immediatelycooled on ice for 5 minutes and then left to reach room temperature inthe HMCKN binding buffer (20 mM Hepes (Sigma), 2 mM MgCl₂ (Sigma), 2 mMCaCl₂ (Sigma), 2 mM KCl (Sigma) and 150 mM NaCl (Sigma), pH 7.4). Thiswas then either incubated with 1590 nM of CFP-10.ESAT-6 heterodimer for1 hour at 37° C. or immediately used in a no protein control which wasdirectly filtered on the nitrocellulose membrane. The ssDNA-proteincomplex was passed through a nitrocellulose membrane. Non-specificallybound ssDNA was removed with two washes of HMCKN binding buffer. BoundssDNA was eluted by cutting the membrane and placing the pieces in aelution buffer (7M urea (Sigma), 100 mM sodium citrate (Sigma) and 3 mMEDTA (Sigma)) and heated to 100° C. for 5 minutes. Thenphenol/chloroform was added and left to incubate for a further 25minutes before extraction. This was followed by chloroform extractionand an ethanol precipitation.

Recovered ssDNA was amplified using polymerase chain reaction (PCR)under mutagenic conditions to increase the diversity of the molecules.This dsDNA was then used in three different methods to generate ssDNA,the three methods were then used as three independent selection methods.The first method was to incorporate a biotin label during PCR using abiotin labelled reverse primer. The dsDNA was then added to streptavidinmagnetic beads (Invitrogen) and the two strands were separated using 0.3M HCl solution or 95° C. for 5 minutes. The resulting ssDNA was cleanedand used as a template for the next round of selection (this method isreferred to as ‘biotin-based selection’). The second method used thedsDNA as a template for in vitro transcription to obtain RNA. The RNAwas DNAse treated, cleaned and precipitated and used as the template forRT PCR to obtain cDNA. The cDNA-RNA complex was treated with a 1 M NaOH(Sigma), 0.5 M EDTA (Sigma) buffer to hydrolyse the RNA. The resultingcDNA was used as a template for the next round of selection (this methodis referred to as ‘T7-based selection’). The third method used to obtainssDNA by including a phosphate labelled reverse primer in the PCR. ThedsDNA was then cleaned up and treated with lambda exonuclease (NewEngland Biolabs, Anatech), this results in the degradation of thereverse strand with the phosphate modification. The resulting ssDNA wasthen cleaned up and used as a template for the next round of selection(this method is referred to as ‘exonuclease-based selection’).

Selection of aptamers against the CFP-10.ESAT-6 heterodimer was doneusing a modified SELEX protocol. Different approaches for partitioningwere used.

A single stranded DNA library was used for the selection of aptamers.After each round of selection using the nitrocellulose membrane forpartitioning, a dsDNA PCR was done to optimise the number of cyclesneeded to convert ssDNA to dsDNA and amplify the selected pool.

Based on the optimisation of the dsDNA PCR the number of cycles giving asingle band at 90 bp was used to produce dsDNA. The optimised number ofcycles was 12 in both selections. The dsDNA was then used to generatethe ssDNA using one of the three methods (biotin, T7- andexonuclease-based selections). The ssDNA obtained from each round wasrun on a native PAGE gel to ensure that the ssDNA obtained was clean foruse in the next round of selection.

The biotin-based selection failed as the two strands could not beseparated and the dsDNA remained bound to the beads. Due to this, thebiotin-based selection was abandoned after the first round of selection.The T7-based selection resulted in an enrichment of 54.7% after 6 roundsof selection and the exonuclease-based selection had an enrichment of68% after 4 rounds of selection. The two pools from the two selectionswere then cloned and sequenced, respectively.

EXAMPLE 2 Cloning and Sequencing of ssDNA Aptamers

The aptamer pool from the 5^(th) (T7-based selection) and 3^(rd)(exonuclease-based selection) SELEX round were subjected to a negativeselection against the nitrocellulose membrane alone. The pools recoveredafter negative selections were put through a final (6^(th)-T7-basedselection and 4^(th)-exonuclease-based selection) SELEX round. In thislast round, both pools of ssDNA were amplified subsequently with thephosphate modified reverse primer and ligated into the pGEM-T easyvector (Promega, Whitehead Scientific). E. coli TOP10 cells (Novagen,Merck) were transformed using these vector constructs. Aftertransformation 244 colonies were picked and spread onto duplicate LBagar (Sigma) plates containing 100 μg/ml of ampicillin (Fermentas,Inqaba Biotech) and IPTG (Fermentas, Inqaba Biotech) for blue whitecolony screening. One plate was used for colony PCR screening using M13primers (IDT, Whitehead Scientific) and to prepare overnight culturesfor glycerol stocks. The other plate was sent to Inqaba Biotech forsequencing with the universal M13 primers (Inqaba Biotech). Sequenceanalysis and alignments were performed using Bioedit. The analysis ofsecondary structure of aptamers was performed by free energyminimisation algorithm according to Zuker using mfold(www.bioinfo.rpi.edu/applications/mfold/).

Of the 244 colonies on the duplicate plates a few were selected forcolony PCR screening using the universal M13 primers to determine ifthey had the insert or not. The T7 selection clones seemed to all havethe insert whereas the exonuclease clones picked for screening wereinsert negative. All clones on the plates were sequenced.

All 244 clones were sent for sequencing and the sequences were analysedusing BioEdit. From the 244 sequences, 104 were insert positive (11 fromthe exonuclease selection and 93 from the T7 selection), this confirmedthe results seen in the PCR screen where most of the exonuclease cloneswere insert negative. Of the 104 sequences that were analysed, 66 wereunique and 15 sequences had two or more repeats, 6 aptamers showedsignificant binding (FIG. 1 and Table 1).

TABLE 1 Sequences (5′-3′ direction) of ssDNA aptamers that significantlybound the CFP-10.ESAT-6 M. tb target antigen

EXAMPLE 3 Binding Assay of ssDNA Aptamers by ELONA

The assay was modified from an ELISA protocols for determination ofaptamer-protein interactions and has been termed an ELONA. Uniqueaptamer clones identified by sequencing were tested for their individualbinding characteristic to the CFP-10.ESAT-6 heterodimer using an ELONA.Each ssDNA aptamer was prepared using the exonuclease method asdescribed above with minor modifications, in that all aptamers wereprepared with the biotinylated forward primer. For each binding assay,96 well micro-titre plates (Corning, Adcock Ingram) were coated with theCFP-10.ESAT-6 heterodimer in a 10 mM NaHCO₃ buffer pH 8.5 (Sigma) andleft overnight at 4° C. The plates were then washed with 1× phosphatebuffered saline containing 0.005% Tween 20 (PBS-T) pH7 (Sigma) andblocked with a 5% fat free milk solution for 1 hour at 4° C. The plateswere then washed 3 times with a 1× PBS after which 150 nM biotinylatedaptamer was added and incubated for 2 hours at room temperature. Thiswas followed by three wash steps with 1× PBS-T and the addition toHRP-conjugated streptavidin (diluted 1:10000 in 1× PBS-T) and incubatedfor two hours at 37° C. The plates were then washed four more times with1× PBS-T, after which a final 3,3′,5,5′-tetramethylbenzidine (TMB)detection substrate (Separations) was added. A change in colour to blue,which could be observed with a naked eye, indicated that the aptamersbound to the CFP-10.ESAT-6 heterodimer. The reaction was stopped with a2 M sulphuric acid solution (Merck), resulting in a colour change fromblue to yellow. The plates were read on the MultiSkan Go plate reader(ThermoScientific, AEC-Amersham) at a wavelength of 450 nm. Each platehad a CFP-10.ESAT-6 and an aptamer alone control, which were averagedand then subtracted from each well to eliminate background noise. Eachaptamer was done in triplicate and the repeats were averaged and thestandard deviation calculated. Each aptamer was tested twice intriplicate to ensure accuracy. The aptamers were then compared to theaptamer alone control using a Student t-test statistical analysis toobtain the p value for significance.

The binding ability of individual aptamers was tested using an ELONA.Out of the 66 biotinylated ssDNA aptamers screened by ELONA against theCFP-10.ESAT-6 heterodimer, 24 bound significantly (p<0.05) (FIG. 2).

EXAMPLE 4 Determination of Antibody Competition Binding of IndividualssDNA Aptamers by ELONA

Only the 24 aptamers that bound significantly to the heterodimer wereused in further studies. The antibody competition binding was done usingthe ELONA method described above with minor modifications. The antibodywas bound to the plate; then blocked followed by the addition of theheterodimer. The biotinylated aptamer was the added, followed by theHRP-conjugated streptavidin. Each combination was done in triplicate andrepeated in two independent assays. The antibody competition data wascompared with the binding assay to identify differences in bindingcapacity of the aptamers in the presence of the antibody. Although thecompetition assay and binding assay were done on different plates, thepositive control (protein and aptamer CSIR 2.11) were run on both plateswith similar results to normalise the data on the two plates.

The positive control ran on both plates was used to normalise theresults between ELONA experiments. Binding of some ssDNA aptamers suchas CSIR 2.15, CSIR 2.9 and CSIR 2.21 was abrogated by the presence ofanti-ESAT-6 monoclonal antibody while binding of other aptamers such asCSIR 2.2 and CSIR 2.12 was enhanced by the presence of the anti-ESAT-6monoclonal antibody (FIG. 3). Taken together, these data suggest thatsome aptamers such as CSIR 2.15, CSIR 2.9 and CSIR 2.21 bind to asimilar epitope on the heterodimer as that recognised by the anti-ESAT-6monoclonal antibody while others such as CSIR 2.2 and CSIR 2.12 bind tomore distant and unique epitopes.

EXAMPLE 5 Determination of Monomer Binding and Specificity of IndividualAptamers by ELONA

Selected ssDNA aptamers were tested for binding specificity to theESAT-6 and CFP-10 monomers. The aptamers were tested for binding usingELONA to CFP-10, ESAT-6, CFP-10.ESAT-6 heterodimer, a CFP-10.ESAT-6related protein in the ESX3 secretion system (EsxGH complex) and a HIVsurface glycoprotein (gp120). EsxGH is an ESAT-6 family related proteinthat is encoded and secreted by the ESX-3 secretion system and gp120 isa HIV glycoprotein which is unrelated to the Mtb antigens.

CSIR 2.11 aptamer was used to test the specificity of the aptamer inrelation to other bacterial lysates. Lysates were obtained by beadbeating 100 ml of cultures of Pseudomonas aeruginosa (Pseudomonas),Streptococcus pyogenes (Streptococcus), Mycobacterium smegmatis(Smegmatis) and the auxotroph of Mycobacterium tuberculosis (Auxotroph).The cutoff for specificity was determined by a 99% confidence intervalof a known negative sample.

The 24 ssDNA aptamers that significantly bind to the heterodimer weretested for monomer binding and specificity. The proteins used forspecificity screening were the monomers (CFP-10 and ESAT-6), theheterodimer (CFP-10.ESAT-6), an ESAT-6 family heterodimer (EsxGH) and anunrelated protein (gp120). While most aptamers specifically bound theCFP-10.ESAT-6 heterodimer, interestingly, two of the 24 aptamers (CSIR2.1 and CSIR 2.12) also bound gp120 to a similar extent or better thanthe CFP-10.ESAT-6 heterodimer (FIG. 4). None of the aptamers were ableto detect EsxGH (FIG. 4). It was also interesting to note that whilemost aptamers also recognised the CFP-10 monomer in addition to theCFP-10.ESAT-6 heterodimer; none of the 24 aptamers screened recognisedthe ESAT-6 monomer (FIG. 4).

Six of the best aptamers were chosen and custom synthesized byIndependent DNA Technologies (IDT) using the solid phase chemicalmanufacturing process. The six aptamers chosen were CSIR 2.2, CSIR 2.9,CSIR 2.15, CSIR 2.19, CSIR 2.21 and CSIR 2.11. The binding of theseaptamers to their CFP-10.ESAT-6 M. tb target proteins, as well as to theCFP-10 and ESAT-6 monomers was repeated to confirm that the aptamerscould be chemically synthesized at an industrial scale with a biotinmodification and still retain their respective activities. Although thereadings of all the synthesized aptamers gave higher readings (FIG. 5)when compared to the in vitro produced aptamers (FIG. 4), there was nosignificant difference between the aptamers synthesized in house by PCRand those custom synthesized by Independent DNA Technologies (IDT) usingthe solid phase process. The results were consistent because all the sixaptamers also recognized the CFP-10.ESAT-6 heterodimer and the CFP-10monomer but not the ESAT-6 monomer (FIG. 5).

CSIR 2.11 was chosen as one of the best aptamers for further specificitytest against other bacterial lysate using the auxotroph strain ofMycobacterium tuberculosis (M. tb) as a positive control. CSIR 2.11 didnot recognise pseudomonas or streptococcus, but recognised the lysate ofMycobacteria smegmatis (FIG. 6). This is not surprising as Mycobacteriasmegmatis also secretes the CFP-10 protein.

EXAMPLE 6 Determination of the Dissociation Constant (K_(D)) of anIndividual ssDNA Aptamer Using the BIAcore 3000

To determine the binding affinity of aptamers to the M. tb antigens, allfour flow cells on a CM5 chip (BIAcore, Separations Scientific) wereactivated with EDC:NHS (BIAcore, Separations Scientific). CFP-10 wasbound to three of the four flow cells by injecting 50 μl of 50 μg/mlCFP-10 over them. Ethanolamine (BIAcore, Separations Scientific) wasthen injected over all four flow cells to quench any remaining activesites. Partially bound or unbound protein was removed by a wash with 10μl of a 10 mM NaOH solution. Different concentrations of the respectiveaptamers (0 nM, 31 nM, 62 nM, 125 nM, 250 nM, and 500 nM) were randomlyinjected over all four flow cells at a flow rate of 10 μl/min for 5minutes. The respective aptamers, were then allowed to dissociate for 10minutes. The flow cell that did not have the aptamer was used as theblank flow cell to subtract non-specific binding. The evaluation wasdone using BiaEvaluation Software (BIAcore) to determine the K_(D)values for each flow cell. The average dissociation constant was thendetermined.

The dissociation constant (K_(D)) of 5 selected aptamers, CSIR 2.2; CSIR2.11; CSIR 2.15; CSIR 2.19 and CSIR 2.19 were determined using theBIAcore surface plasmon resonance technology. CSIR 2.19 had the lowestK_(D) at 1.6±0.5 nM, while CSIR 2.11 had a K_(D) of 8±1.07 nM and CSIR2.2 had comparatively the highest K_(D) at 21.5±4.3 nM (FIG. 7).

EXAMPLE 7 Determination of whether Aptamer Folding Affects the Bindingto the Target

It was important to determine if the aptamers needed to be refolded forfurther studies as this would impact on their downstream application.One batch of CSIR 2.11 aptamer was folded as described above whileanother batch was used directly after thawing to determine if theaptamer could be used without the refolding step. An ELONA was preformedwith both batches of aptamer against CFP-10. The results show that theaptamer can be used directly from the freezer without the refolding step(FIG. 8).

EXAMPLE 8 Limit of Detection

CFP-10 was bound to a 96 well plate (Corning, Adcock Ingram) in serialdilutions in a NaHCO₃ buffer and left overnight at 4° C. The plates werethen washed with 1× phosphate buffer saline solution containing 0.005%Tween 20 (PBS-T) pH7 (Sigma) and blocked with a 5% fat free milksolution for 1 hour at 4° C. The plates were then washed 3 times with a1× PBS-T after which 150 nM biotinylated EA10 aptamer was added andincubated for 2 hours at room temperature. This was followed by threewash steps with 1× PBS-T and the addition of HRP-conjugated streptavidin(diluted 1:10000 in 1× PBS-T) and incubated for two hours at 37° C. Theplates were then washed four more times with 1× PBS-T, after which the3,3′,5,5′-tetramethylbenzidine (TMB) detection substrate (Separations)was added. A change in colour to blue, which could be observed with anaked eye, indicated that the aptamers bound to the CFP-10 protein. Thereaction was stop with a 2 M sulphuric acid solution (Merck), at whichpoint the blue colour observed changes to yellow. The plates were thenread on the MultiSkan Go plate reader (ThermoScientific, AEC-Amersham)at a wavelength of 450 nm. Each plate had a CFP-10 and an aptamer alonecontrol, which were averaged and then subtracted from each well toeliminate background noise. Each dilution was done in triplicate, therepeats averaged and the standard deviation calculated.

Serial dilutions of CFP-10 coated on a 96 well micro-titer plate showedthat 150 nM of CSIR 2.11 aptamer is able to detect as little as 31 ng ofCFP-10 (FIG. 9). The best fit curve had an R² value of 0.85.

EXAMPLE 9 Evaluation of Clinical Samples from Patients with or withoutActive TB

Twenty sputum samples that had been well characterized (Table 2) wereused. Briefly, they included: (a) smear positive—culture positive; (b)smear negative—culture positive; (c) smear negative—culture negative,quantiferon positive and TSPOT positive; and (d) smear negative—culturenegative, quantiferon negative and TSPOT negative. These sputum sampleshad been liquefied in a 0.1% dithiothreitol (DTT) solution. The sputumsamples were bound to a 96 well micro-titer plate (Corning, AdcockIngram) in a NaHCO₃ buffer and left overnight at 4° C. The plates werethen washed with 1× PBS-T pH7 and blocked with a 5% fat free milksolution for 1 hour at 4° C. The plates were then washed 3 times with a1× PBS-T after which 300 nM biotinylated EA10 aptamer was added andincubated for 2 hours at room temperature. This was followed by threewash steps with 1× PBS-T and the addition of HRP-conjugated streptavidin(diluted 1:10000 in 1× PBS-T) and incubated for two hours at 37° C. Theplates were washed four more times with 1× PBS-T, after which a finalTMB (Separations) detection substrate was added. A change in colour toblue, which could be observed with a naked eye, indicated that theaptamers bound to the CFP-10 protein. The reaction was stopped with a 2M sulphuric acid solution (Merck) at which point the blue colourobserved changed to yellow. The plates were then read on the MultiSkanGo plate reader (ThermoScientific, AEC-Amersham) at a wavelength of 450nm. Each plate had an aptamer alone control, which were averaged andthen subtracted from each well to eliminate background noise. Eachsample was done in triplicate, averaged and the standard deviation wascalculated; the experiment was repeated twice to determine repeatabilityof the assay. An aptamer selected from the same library against adifferent target was used as a control (CSIR 3.13). The cutoff forspecificity was determined by a 99% confidence interval of a knownnegative sample.

TABLE 2 Characteristics of sputum samples obtained from patients with orwithout active TB. Based on the results of tests used to characterisethe samples, the samples were broadly classified as No TB; Latent TB; orActive TB as denoted in the parenthesis. Numbers were assigned to thesamples during the study in order to match the patients' numbers. Smearnegative - Smear negative - culture negative, culture negative,quantiferon quantiferon Smear negative and positive and negative - Smearpositive - TSPOT negative TSPOT positive culture positive culturepositive (No TB) (Latent TB) (Active TB) (Active TB) 35 54 11 5 52 66 3617 139 67 65 39 143 76 85 59 161 101 88 94

The CSIR 2.11 ssDNA aptamer was able to accurately detect 4 of the 5smear positive—culture positive samples as positive (80%), 4 of the 5smear negative—culture positive samples as positive (80%); but it alsodetected one sample classified as no TB (sample 139, which is allnegative for quantiferon, TSPOT, smear and culture tests), as positive(FIG. 10). In addition, CSIR 2.11 detected 3 of the 5 samples classifiedas latent TB (quantiferon and TSPOT positive but smear and culturenegative) as positive (FIG. 14). The control ssDNA aptamer, CSIR 3.13,which was derived from the same parental library but isolated against,and specific for human CD7 was negative for all the samples, as expected(data shown for only 1 sample on FIG. 10). The cut-off for negativeresults using the aptamers was determined to be an OD₄₅₀ below 0.2 at99% confidence interval based on a known negative sample (FIG. 10).Taken together, and based on the classification for latent TB used incharacterizing the samples, CSIR 2.11 ssDNA aptamer had a specificity of60% (i.e. correctly identified 6/10 samples as negative) and asensitivity of 80% (i.e. correctly identified 8/10 samples as positive)using the ELONA readout platform. Notwithstanding, two of the falsenegatives samples in this study (samples 36 and 59) were inconclusivebecause they were on the border line of the cut-off (FIG. 10). If theywere taken as positive the sensitivity of CSIR 2.11 would increase from80% to 100%.

Evaluation of 80 sputum samples using CSIR 2.11 as detection reagent wasconducted, the results of which are illustrated in FIG. 11. The aptamerwas tested on three groups of samples (A) Definite TB, (B) Latent TB andTB negative and (C) healthy laboratory volunteers. Using Youden's index,the cut-point for positive samples was set at an OD450 of 0.2 and isindicated by the dotted line. Data are presented as mean±standarddeviation of the mean.

Current diagnostics for TB have many disadvantages, especially when usedin resource-poor settings, which also happen to be high in TB incidenceand prevalence. The current gold standard for TB diagnostics is acombination of smear microscopy and culture methods. The advantage ofsmear microscopy is the relatively low cost. The main disadvantage isthat smear microscopy has low sensitivity (35-75%), especially among HIVpositive patients. While the culture method increases sensitivity toover 90%, it takes 6-8 weeks to get results and the method also requireshighly trained personnel and specialized containment level 3 facilities.The culture method currently costs about $10 per sample, excluding theexorbitant costs of establishing and maintaining a containment level 3laboratory. There seem to be a correlation between the duration of test,cost of test and sensitivity. Serological tests are rapid and relativelyinexpensive but have poor sensitivity (16-75%), NAATs are rapid but onlyhave a sensitivity of 60-70%. The GeneXpert® is a fully automatedmolecular test with a sensitivity of 60-80% but is currently not a costeffective method for poor resource settings. It currently costs about$20 USD per sample, excluding the high price of the instrument. Despitecurrent achievements, there is still a need for an Affordable,Sensitive, Specific, User-friendly, Rapid, Equipment-free andDeliverable to end user (ASSURED) TB diagnostics.

DNA aptamers by virtue of their simplicity, specificity, sensitivity andlow costs of production can serve as ASSURED TB diagnostics, thusmeeting the needs of a diagnostic tool that is required inunderdeveloped and high burden TB countries. In a currentproof-of-concept study, using the ELONA readout platform, we showed thata single stranded DNA aptamer called CSIR 2.11 isolated against theCFP-10.ESAT-6 Mycobacterium tuberculosis target protein can detect TB inwell characterized clinical sputum samples of TB patients from a highHIV prevalence country with a sensitivity of 80-100% and specificity of60% if latent TB is considered negative.

The two false negative readings are on the border line of the cut-offand the results are thus inconclusive. They could be classed as eitherpositive or negative. The reason for the strikingly false positivesample (139) is hard to find, unless the sample was cross-contaminatedduring the process of acquiring sputum and/or during sample preparationand storage.

The low cost of $0.52 USD (R3.67 ZAR) per sample for the aptamer-basedELONA for TB detection and the rest of data in general, including the80-100% sensitivity, demonstrate that the aptamers of the presentinvention can be used successfully and economically in ASSURED TBdiagnostics.

EXAMPLE 10

Further characterisation and optimisation of the anti-ESAT-6.CFP-10aptamers yielded active, affordable TB detection molecules for thepotential development of a PoC TB diagnostic tool.

Rational truncation of the original sequences yielded shorter, lowercost molecules that show comparable activity to the original parentsequences. The truncated versions of the aptamers retained the parts ofthe original (parent) aptamers predicted to play a role intarget-binding as can be predicted through secondary structures

Truncation of aptamer CSIR 2.11 was effected by secondary structure(2D)-guided methods. The predicted 2D structure for the originalsequence of aptamer CSIR 2.11 consists of three stem-loops. Truncation,T2, (77-mer) resulted from cutting out 12 nucleotide bases in thedirection 3′ to 5′. All the stem-loops, along with their assignedminimum free energy, are all retained in the predicted 2D structures ofboth truncated versions of the aptamer.

Truncation of aptamer CSIR 2.19 was effected by secondary structure(2D)-guided methods. The predicted 2D structure for the originalsequence of aptamer CSIR 2.19 consists of two stem-loops. CSIR 2.19Truncated (77-mer) resulted from cutting out 12 nucleotide bases in thedirection 3′ to 5′. Both stem-loops, along with their assigned minimumfree energy values, are all retained in the predicted 2D structures ofboth truncated versions of the aptamer.

The truncated aptamers show binding to target proteins, with affinitiescomparable to those of the parent sequences (FIG. 12).

Binding of full length and truncated aptamers to the ESAT-6/CFP-10 dimerand CFP-10 was assessed by ELONA.

FIG. 12A shows apparent binding of CSIR 2.11: the truncated 77-mer boundto the target proteins with affinity values comparable to those of theoriginal 90-mer aptamer.

FIG. 12B shows apparent binding of CSIR 2.19 aptamer: the 77-mer boundto the target proteins with affinity values comparable to those of theoriginal 90-mer aptamer for both ESAT-6/CFP-10 dimer and the CFP-10monomer. The 77-mer showed slightly higher affinity (*P-value<0.05) forthe ESAT-6/CFP-10 dimer than did the 90-mer.

The truncated aptamers can be used in an aptamer-based TB diagnostictool.

EXAMPLE 11

The original full length aptamers can be used in pairs for further usein a diagnostic setting. To test the aptamers in a sandwich ELONA, 96well micro-titre plates (Corning, Adcock Ingram) were coated with theCSIR2.11 in a 10 mM NaHCO₃ buffer pH 8.5 (Sigma) and left overnight at4° C. The plates were then washed with 1× phosphate buffered salinecontaining 0.005% Tween 20 (PBS-T) pH7 (Sigma) and blocked with a 5% fatfree milk solution for 1 hour at 4° C. The plates were then washed 3times with a 1× PBS-T after which 500 ng of CFP-10.ESAT-6 heterodimerwas added and incubated for 2 hours at room temperature. This wasfollowed by three wash steps with 1× PBS-T and the addition of 150 nMbiotinylated aptamer and incubated for a further two hours at roomtemperature. Following this the plate was washed 3 times with a 1× PBS-Tafter which a HRP-conjugated streptavidin (diluted 1:10000 in 1×PBS-T)was added and incubated for two hours at 37° C. The plates were thenwashed four more times with 1× PBS-T, after which a final3,3′,5,5′-tetramethylbenzidine (TMB) detection substrate (Separations)was added. A change in colour to blue, which could be observed with anaked eye, indicated that the aptamers function in a sandwich format.The reaction was stopped with a 2 M sulphuric acid solution (Merck),resulting in a colour change from blue to yellow. The plates were readon the MultiSkan Go plate reader (ThermoScientific, AEC-Amersham) at awavelength of 450 nm. The pairs were then compared to the no proteinpair control using a Student t-test statistical analysis to obtain the pvalue for significance.

Several potential pairs have been identified, as shown in FIG. 13.

EXAMPLE 12

Evaluation of sputum samples using CSIR 2.21 as detection reagent wasconducted and compared to the results using CSIR 2.11. A comparison ofthe ability of CSIR 2.21 and CSIR 2.11 was performed on 28 sputumsamples to evaluate the use of CSIR 2.21 in a clinical setting. Thecomparison was done using an ELONA as in Example 9 using either CSIR2.11 or CSIR 2.21 as a detection molecule. The comparison is illustratedby FIG. 14 and indicates that both aptamers yielded similar results. Asillustrated by FIG. 14, the sensitivity and specificity of CSIR 2.11 anda more specific aptamer, CSIR 2.21, were compared using 28 sputumsamples. Using Youden's index, the cut-point for positive samples wasset at an OD₄₅₀ of 0.1 and is indicated by the solid line. Data arepresented as means±standard deviation of the mean. Samples that gave apositive result when using CSIR 2.21 as a detection molecule are denotedas CSIR 2.21 positive, while negative samples are denoted as CSIR 2.21negative. Samples that gave a positive result when using CSIR 2.11 as adetection molecule are denoted as CSIR 2.11 positive, while negativesamples are denoted CSIR 2.11 negative.

EXAMPLE 13

The aptamers were tested for the detection of whole bacteria, which isuseful for diagnostics. To evaluate the aptamers detection of wholebacteria, an ELONA similar to that described in Example 9 was performed,except instead of coating with sputum samples the plate was coated withMTB culture at an OD₆₀₀=1, OD₆₀₀=0.5 and OD₆₀₀=0.25. The whole bacteriaELONA experiments were conducted using the six aptamers: CSIR 2.11, CSIR2.19, CSIR 2.21, CSIR 2.15, CSIR 2.2 and CSIR 2.9. All six of theaptamers were all able to detect whole bacteria as illustrated in FIG.15.

While the invention has been described in detail with respect tospecific embodiments thereof, it will be appreciated that those skilledin the art, upon attaining an understanding of the foregoing may readilyconceive of alterations to, variations of and equivalents to theseembodiments. Accordingly, the scope of the present invention should beassessed as that of the appended claims and any equivalents thereto.

1-14. (canceled)
 15. An oligonucleotide which is a DNA aptamer, or apairing of two DNA aptamers, that binds to a CFP-10.ESAT-6 heterodimeror to a CFP-10 monomer of a Mycobacterium strain.
 16. Theoligonucleotide of claim 15 which is selected from the group comprisingSEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5 andSEQ ID NO: 6, a complementary oligonucleotide thereof, anoligonucleotide being at least 80% homologous thereto, or a pairing ofany of the aforementioned.
 17. An in vitro method of diagnosingtuberculosis (TB), said method comprising: (a) contacting a sample takenfrom an individual suspected to be infected with TB with theoligonucleotide of claim 15 in a CFP-10.ESAT-6 heterodimer bindingassay; and (b) determining whether or not the oligonucleotide has boundto a CFP-10.ESAT-6 heterodimer in the sample, with binding of theoligonucleotide to the CFP-10.ESAT-6 heterodimer thus confirming thepresence of the CFP-10.ESAT-6 heterodimer, and hence TB infection in thesample.
 18. An in vitro method of diagnosing tuberculosis (TB), saidmethod comprising: (a) contacting a sample taken from an individualsuspected to be infected with active TB, or latent TB, with theoligonucleotide of claim 15 in a CFP-10 monomer binding assay; and (b)determining whether or not the oligonucleotide has bound to a CFP-10monomer in the sample, with binding of the oligonucleotide to the CFP-10monomer thus confirming the presence of the CFP-10 monomer, and hence TBinfection in the sample.
 19. The method of claim 17, wherein the bindingassay is a modified ELISA-type assay, wherein the antibodies against theCFP-10.ESAT-6 heterodimer or the CFP-10 monomer are replaced by theoligonucleotide of claim
 15. 20. The method of claim 17, wherein theoligonucleotide is that of SEQ ID NO:
 3. 21. A diagnostic kit fordiagnosing tuberculosis (TB), said kit including: (a) a device fortaking a sample from an individual suspected to be infected with TB; (b)apparatus for applying the method of diagnosing TB according to claim17; (c) the oligonucleotide of claim 15; and (d) optionally, a positivecontrol and/or a negative control.
 22. The method of claim 18, whereinthe binding assay is a modified ELISA-type assay, wherein the antibodiesagainst the CFP-10.ESAT-6 heterodimer or the CFP-10 monomer are replacedby the oligonucleotide of claim
 15. 23. The method of claim 18, whereinthe oligonucleotide is that of SEQ ID NO:
 3. 24. A diagnostic kit fordiagnosing tuberculosis (TB), said kit including: (a) a device fortaking a sample from an individual suspected to be infected with TB; (b)apparatus for applying the method of diagnosing TB according to claim18; (c) the oligonucleotide of claim 15; and (d) optionally, a positivecontrol and/or a negative control.